daravuth cheam dee denver, p h d the denver lab department of zoology hhmi summer 2011
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HHMI Project 2011 Investigating Possible Cryptic Species of Xiphinema Nematodes through DNA Analysis. Daravuth Cheam Dee Denver, P h D The Denver lab Department of Zoology HHMI Summer 2011. Introduction. - PowerPoint PPT PresentationTRANSCRIPT
DARAVUTH CHEAMDEE DENVER, PhDTHE DENVER LAB
DEPARTMENT OF ZOOLOGYHHMI SUMMER 2011
HHMI Project 2011Investigating Possible Cryptic Species of
Xiphinema Nematodes through DNA Analysis
INVESTIGATING POSSIBLE CRYPTIC SPECIES OF XIPHINEMA NEMATODES
THROUGH MITOCHONDRIAL GENOMIC ANALYSIS
Introduction
Introduction
Xiphinema americanumo Plant parasitic nematode.o Can vector viruses such as the Tomato Ringspot Virus.
Infects tomatoes, berries, grapes, etc.o Both the nematode itself and the virus are detrimental
to agriculture. Causes millions of dollars of damage.
Mitochondrial Genome (mtDNA)
18S Ribosomal DNA(nDNA)
SmallCircularMaternally
inheritedUsually doesn’t
undergo genetic recombination
Undergoes fast evolutionary changes
Used to identify an organism’s taxonomic group.
LinearBiparentally
inheritedSlow evolving.
Introduction
X. americanum Mitochondrial Genome
Click icon to add picture Small
mitochondrial genome.o Smaller rRNA
genes.o Smaller tRNA
genes. Missing five
tRNA genes Missing a
conserved noncoding region.
Taken fromHe et al. 2005
X. americanum Mitochondrial Genome
Introduction
Click icon to add picture
mtDNA: A Good Genetic Marker
Introduction
mtDNA is used to define and differentiate species.o Comes into
conflict with morphology.
Similar morphology, but different genetic make-up entails a cryptic a species.
What defines a species?
Morphology DNAVs.
IntroductionIntroduction
Similar Morphology
Different DNA Sequences+ =
Cryptic Species
Cryptic Species:What & Why
mtDNA: ≥ 90.0% SimilarnDNA: ≥ 98.5% Similar
Hypothesis
X. americanum
Introduction
• X. americanum may contain cryptic species.
• PCR amplify and sequence DNA of multiple samples of presumed X. americanum nematodes.
Species I Species II Species III
INVESTIGATING POSSIBLE CRYPTIC SPECIES OF XIPHINEMA NEMATODES
THROUGH MITOCHONDRIAL GENOMIC ANALYSIS
Materials & Methods
Materials & Methods
EQ FMN4C2
PEN10066-3
C027PL
C037A1
NWABNC2
OHIO2
DINNPST213
Materials & Methods
Location of Origin ZL Number Host Virus (+/-)
Pennsylvania PEN1 Grape ToRS
South West Washington 0066-3 Blueberry ToRS
Colorado CO27PL Cherry Cherry Rasp Leaf
CO37A1 Cherry Cherry Rasp Leaf
Arkansas NWAB Blackberry TRS
North Carolina NC2 Blackberry TRS
Oregon EQ Grape ToRS
FM Grape -
N4C2 Grape -
Ohio OHIO2 Blueberry -
Eastern Washington DINNP Grape -
ST213 Grape -
Population Isolates
Mitochondrial Genome:Procedure
18S Ribosomal DNA:Procedure
Materials & Methods
PCR Technique
DNASequencing
Gel Electro-phoresis
BLAST Verification
PCR Technique
DNA Alignment
DNASequencing
BLAST Verification
Mitochondrial Genome:PCR Amplicon
18S Ribosomal DNA:PCR Amplicon
Materials & Methods
Taken fromHe et al. 2005
18S 5.8S 28S
18S Ribosomal DNA
Genetic Source: nDNA # of Amplicons: 1 Length: ~900 bp
Mitochondrial Genome
Genetic Source: mtDNA # of Amplicons: 2 Length: ~12,626 bp
INVESTIGATING POSSIBLE CRYPTIC SPECIES OF XIPHINEMA NEMATODES
THROUGH MITOCHONDRIAL GENOMIC ANALYSIS
Results & Discussion
Mitchochondrial Genome: DNA
Sequences
Mitochondrial Genome:Phylogeny Tree (7203
bp)
Results & Discussion
88% Match
99% Match
80% Match
18S Ribosomal DNA:DNA Sequences
18S Ribosomal DNA:Phylogeny Tree (541 bp)
Results & DiscussionResults & Discussion
99 - 100% Match
99 - 100% Match
Mitochondrial Genome:DNA Sequences
18S Ribosomal DNA:DNA Sequences
Results & Discussion
Mitochondrial Genome:Phylogeny Tree (7203
bp)
18S Ribosomal DNA:Phylogeny Tree (541 bp)
Results & Discussion
Results & Discussion
COI Gene Phylogeny Tree (335 bp)
INVESTIGATING POSSIBLE CRYPTIC SPECIES OF XIPHINEMA NEMATODES
THROUGH MITOCHONDRIAL GENOMIC ANALYSIS
Conclusions
Click icon to add pictureX. americanum
Species I Species II Species III
General Phylogeny of Three Cryptic Species
According to nDNA, there aren’t cryptic species.
According to mtDNA, there are cryptic species.o Possibly
three different kinds.
Conclusions
OHIO2NC2NWABC027PLC037ADINNPEQFMN4C2
PEN10066-3
ST213
MitochondrialDNA Analyses
NuclearDNA Analyses
Search for a region of DNA sequences to be used as a diagnostic locus.
o COI gene.
Sequence whole 18S rDNA.
Sequence other parts of nDNA to find any differences.
Future WorkConclusions
Acknowledgements
The Denver LabStaff Dee Denver, PhD Katie Clark, PhD Dana Howe, MSStudents Michael Raboin, PhD Samantha Colby, BS Kristin Gafner, BS Jonathan Seng, BS Sita Ping, BSHorticultural Crops Research Lab Inga Zasada, PhD, USDA-ARS
HHMI Summer Research Program
Kevin Ahern, PhD Dan Arp, PhDCripps Scholarship FundDepartment of Biochemistry
andBiophysics Dina Stoneman, Office
Specialist
Questions?
?